PONE-D-20-18593

Prognostic Significance of BIRC7/Livin, Bcl-2, p53, Annexin V, PD-L1, DARC, MSH2 and PMS2 in Colorectal Cancer Treated with FOLFOX Chemotherapy with or without Aspirin

PLOS ONE

Dear Dr. Faruk,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The manuscript should be edited for scientific clarity, including providing a clear rationale.

Additional details have to be provided on the aspirin trial.

Please submit your revised manuscript by Oct 30 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Irina V. Lebedeva, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: A mountain of work presented here impressively presented with interpretations provided

It would be helpful to have more details of the 92 colorectal cancers in the aspirin trial - age, tumour location in the bowel, stage, microsatellite stability in view of the ASPREE study, were there any age related effects on the biomarker outcomes?

Was there a differential effect of aspirin seen in the MSS vs MSI-H patients or models?

Reviewer #2: PLOS ONE

Prognostic Significance of BIRC7/Livin, Bcl-2, p53, Annexin V, PD-L1, DARC, MSH2 and PMS2 in Colorectal Cancer Treated with FOLFOX Chemotherapy with or without Aspirin

The authors investigate several players in the apoptotic cascade (pro and anti-apoptotic markers) in CRC, with a special focus on the African population.

The authors have 2 main approaches- patients and a CRC rat model and obrain several interesting results.

However I have several major concerns, but I believe authors will be able to answer accordingly. The results are not well presented-dispersed through several pages (same figure), also lack N etc, making it very hard to read think and analyse the data- I propose several improvements to help the reader. Also the rational is not well explained or even justified – making it hard to follow the rational, also lacks subtitles of the results also making it very hard to follow so many data.

ABSTRACT

3rd sentence of the abstract – is confusing or misleading ….

“Various cellular processes are associated with evasion of apoptosis. These include overexpression of pro-apoptotic and anti-apoptotic proteins (including BIRC7/Livin, Bcl-2, p53, Annexin V, PD-L1, and DARC) and dysregulation of DNA mismatch repair proteins (including MSH2 and PMS2).” this confusing because

BIRC7/Livin, Bcl-2 are anti-Apoptotic; p53 – pro-apoptotic ; PDL-1- can induce apoptosis and DARC- XXX

AnexinV is a marker not really a pro-apoptotic protein

So everything is put in the same bag and becomes very confusing..and many time the rational is lost.

So please re-write this sentence.

INTRODUCTION

Again concepts are mixed and confusing -please clarify .

1. I am not an expert on the apoptotic pathway etc but what I read was that BIRC7/Livin is an inhibitor of caspases – an anti-apoptotic protein and in the following sentences is stated this but also the opposite…so I don’t understand…:

Please clarify

“Thus, BIRC7/Livin serves as executioner of caspase-3 and-7 and also as an initiator of caspase-9 via the BIR domain (16). The BIR domain interacts with caspases active site to promote the degradation of active caspases (17). Apoptosis occurs when proteolytic activation of the cysteine-dependent 3 aspartate-directed proteases family (caspases) initiates and cleaves to effector caspases for the breakdown of intracellular protein substrates (18). BIRC7/Livin interacts with the second mitochondria-derived activator of caspases (SMAC) through the BIR domain to promote the caspase

activation in the cytochrome c pathway (13, 16)”.

2. Also needs revision:

The p53 protein is important for initiation of the apoptotic stimuli during anticancer therapy by

cleaving to the sites of damage in the cellular DNA (22). Thus, under normal cellular conditions, the p53 transcription factor regulates various cell functions such as, apoptosis activation and control of cell growth, migration, and invasion (23

P53 to my knowledge is able sense DNA damage and activate a series of cellular processes such as arrest cell cycle and induce apoptosis… not “cleaving to the sites of damage in the cellular DNA”

3. Also the role for AnexinV is not clear – Anexin5 can be used as a marker to detect apoptotic cells by its ability to bind to phosphatidylserine (outer leaflet of the plasma membrane). The protein per se, I believe the function is not yet very clear and has been highly correlated with coagulation and platelets – so I do wonder if the upregulation detected in the experiments Is not due to the TME and some platelet reaction…I think the authors should explain better the state of the art what is known and the different hypothesis..

4. Aspirin rational is not fully explained …confusing sentences… to my knowledge Aspirin by inhibiting COX can lead to an innate immune de-repression

“Aspirin inhibits cyclooxygenase (COX) enzymes (COX-1 and COX-2) -which are upregulated at sites of inflammation, to facilitate apoptosis (37).” Who facilitates apoptosis – aspirin? Or COX?

To my knowledge aspirin may work by inhibiting COX, reducing PGE2 production which induces a pro-tumor inflammatory profile and aspirin can revert this towards a classic anti-cancer immune pathway – therefore aspriirn could be used as adjuvant for immune checkpoint therapy (PMC4597191; DOI: 10.1016/j.cell.2015.08.015)

RESULTS

1. Figures are very difficult to follow with panels from the same figure spanning several pages ! Please put all panels in one figure A4 with the corresponding legend.

2. I could not find the N- number of patients analysed, number of models or number of independent experiments in all figures

3. Please put all pictures with the same magnification – also allows a better reading and analysis of the results

4. When possible please do dot charts – each dot a patient/ or mouse so we can analyse the dispersion and also number of data points

5. Please introduce subtitles of results

6. Could not find Table 3

7. It would be very important to show in your patient data – the correlation between the expression patern of the different markers and response to the neoadjuvant treatments – to understand for instance in Fig 3 the increase of BIRC7 in mucinous adenocarcinoma group upon FOLFOX treatment occurred in the ones that responded well or the poor responders? Within the good responders – they still have residual disease? Is this upregulation real or is just the escapers resistant clones that express high antiapoptotic proteins? the N is not specified …

8. Not clear when is adjuvant or neoadjuvant FO treatment

9. Fig. 6 why is FAcid only shown in I – also no comment why FA can induce so many effects…

10. Fig7 – results confusing sometimes you use anexin as a readout of apoptosis others not – but if you are using an antibody against Anexin is not the same thing as using anexin5 staining …confusing please clarify

11. Also all data of the rat CRC – could not see the impact of treatment on tumor progression – maybe I missed but it would be very important to correlate all results to response to treatment

DISCUSSION

“Cancer chemotherapeutics such as FOLFOX may induce apoptosis by targeting anti

apoptotic genetic mutations and equally reduce drug sensitivity which may lead to treatment failure

(38, 46).”

To my knowledge FOLFOX CT is thought to induce direct damage to cancer cells – DNA damage /cytotic damage etc that then induces apoptosis not targeting “apoptotic genetic mutations…”

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6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-20-18593R1

Prognostic Significance of BIRC7/Livin, Bcl-2, p53, Annexin V, PD-L1, DARC, MSH2 and PMS2 in Colorectal Cancer Treated with FOLFOX Chemotherapy with or without Aspirin

PLOS ONE

Dear Dr. Faruk,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please address the issues indicated by the Reviewer 2.

Please submit your revised manuscript by Jan 28 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Irina V. Lebedeva, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Well done! I think the authors have been diligent and presented a comprehensive range of data supporting their claim - which is an important one clincally

Reviewer #2: Overall I do not think the authors answer to all my concerns...

1-The sentence in the third line of the abstract was re-written as follows:

These include overexpression of pro-apoptotic and anti-apoptotic proteins (including p53 and PD-L1; BIRC7/Livin and Bcl-2), chemokine receptors (including DARC), and dysregulation of DNA mismatch repair proteins (including MSH2 and PMS2).

This continues confusing should put in separated brackets each class of genes

2- In the discussion, the sentence was re-written as follows:

Cancer chemotherapeutics such as FOLFOX may induce direct damage to cancer cells with increase apoptosis and equally reduce drug sensitivity which may lead to treatment failure.

Please clarify -what do the authors want to say? What do you mean by FOLFOX reducing drug sensitivity? That after FOLFOX treatment resistant clones emerge?

3- Could not find a clarification of why the rational for testing aspirin in the first place-was one of my concerns and i could not find where it was answered.

4-The figures continue in a very strange format - do not understand why Fig3 -is dispersed in 4 slides! fig4 in 3 slides???

5- no dot plots as requested

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Finlay Macrae

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Prognostic Significance of BIRC7/Livin, Bcl-2, p53, Annexin V, PD-L1, DARC, MSH2 and PMS2 in Colorectal Cancer Treated with FOLFOX Chemotherapy with or without Aspirin

PONE-D-20-18593R2

Dear Dr. Faruk,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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Kind regards,

Irina V. Lebedeva, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No